(a) Field of the Invention
The invention relates to a novel enzyme, DNA demethylase, therapeutic and diagnostic uses thereof.
(b) Description of Prior Art
Many lines of evidence have established that modification of cytosine moieties residing in the dinucleotide sequence CpG in vertebrate genomes is involved in regulating a number of genome functions such as parental imprinting, X-inactivation, suppression of methylation of ectopic genes and differential gene expression (Szyf, M. (1996) Pharmacol. Ther. 70, 1–37). DNA methylation performs its function of differentially marking genes because the distribution of methylated CpGs is tissue- and site-specific forming a pattern of methylation (Szyf, M. (1996) Pharmacol. Ther. 70, 1–37). It is clear that the pattern of methylation is fashioned by a sequence of methylation and demethylation events (Brandeis, M. et al. (1993) Bioassays 15, 709–713) during development and is maintained in the fully differentiated cell (Razin, A. et al. (1980) Science 210, 604–610). While it was originally suggested that DNA demethylation is accomplished by a passive loss of methyl groups during replication (Razin, A. et al. (1980) Science 210, 604–610), it is now clear that an active process of demethylation occurs in embryonal cells (Frank, D. et al. (1991) Nature 351, 239–241), in differentiating cell lines (Razin, A. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2827–2831; Szyf, M. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8090–8094) and in response to estrogen treatment (Saluz, H. P. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 7167–7171). Two modes of demethylation have been documented: site specific demethylation that coincides in many instances with onset of gene expression of specific genes and a general genome wide demethylation that occurs during early development in vivo during cellular differentiation and in cancer cells (Feinberg, A. P. et al. (1983) Nature 301, 89–92; Razin, A. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2827–2831). The global demethylation is consistent with the hypothesis that a general demethylase activity which is activated at specific points in development or oncogenesis exists. It has been hypothesized that one mechanism regulating the pattern of methylation is the control of expression of methyltransferase (Szyf, M. (1991) Biochem. Cell Biol. 69, 764–767) and demethylase activities (Szyf, M. (1994) Trends Pharmacol. Sci. 7, 233–238). Although extensive information has been obtained on the enzymatic activity responsible for methylation and the regulation of its expression in the last two decades (Szyf, M. (1996) Pharmacol. Ther. 70, 1–37), the identity of the demethylase has remained a mystery. It is clear however that to fully understand how patterns of methylation are formed and maintained and to determine their role in development, physiology and oncogenesis, one has to identify the demethylase enzyme(s). Two main difficulties have inhibited the identification of this enzyme. First, it is believed that demethylation of a methylated cytosine is chemically highly unlikely since it involves breaking a very stable C—C bond. Second, demethylation occurs at very defined stages in development (Brandeis, M. et al. (1993) Bioassays 15, 709–713) and identifying an adequate tissue source for this enzyme is critical.
Whereas no bona fide demethylase has been identified to date, alternative biochemical mechanisms involving exchange of methylated cytosines with non-methylated cytosines have been described. One previously proposed mechanism is removal of the methylated base by a glycosylase and its replacement with a non-methylated nucleotide utilizing an “excision-repair” mechanism (Razin, A. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2827–2831). Glycosylase activities that can remove methylated cytosines from DNA have been demonstrated by Vairapandi and Duker (Vairapandi, M. et al. (1993) Nucl. Acids Res. 21, 5323–5327) and more recently by Jost (Jost, J. P. et al. (1995) J. Biol. Chem. 270, 9734–9739). However it is not clear whether this activity is responsible for the general demethylation observed in cellular differentiation. The fact that the activity identified by Jost acts specifically on hemimethylated sequences (which is not the natural substrate in most cases) and can remove thymidines as well as 5-methylcytosines, supports a repair function for this glycosylase-demethylase (Jost, J. P. et al. (1995) J. Biol. Chem. 270, 9734–9739). An alternative mechanism involving a RNA dependent activity has been recently described by Weiss et al. (Weiss et al., 1996). This proteinase-insensitive RNA dependent activity has been shown to catalyze the excision and replacement of a methylated CpG dinucleotide with a nonmethylated CpG dinucleotide that is contained in a DNA-RNA hybrid molecule (Weiss, A. et al. (1996) Cell 87, 709–718). This activity which was identified in differentiating cells in culture was proposed to be involved in demethylation during development. These previous findings demonstrate that the common accepted model in the filed has been that a bona fide demethylase does not exist.
It has been previously proposed that the extensive hypomethylation observed in cancer cells might be a consequence of activation of demethylase activity by oncogenic pathways (Szyf, M. (1994) Trends Pharmacol. Sci. 7, 233–238; Szyf, M. et al. (1995) J. Biol. Chem. 270, 12690–12696). In accordance with this hypothesis we have shown that ectopic expression of v-Ha-ras had induced demethylation activity in the cells (Szyf, M. et al. (1995) J. Biol. Chem. 270, 12690–12696). Using an assay that directly measures the conversion of 3′ 32P labeled methyl dCMP (mdCMP) into dCMP, we have shown that nuclear extracts prepared from P19-Ras transfectants bear high levels of demethylase activity (Szyf, M. et al. (1995) J. Biol. Chem. 270, 12690–12696). Building on this observation, we hypothesized that cancer cell lines were a good source for demethylase. However, it is not evident that Ras expression in p19 cells does reflect the situation in cancer cells. P19 is an embryonic cell and expression of Ras might be differentiating them.
It would be highly desirable to be provided with a bona fide DNA demethylase (DNA dMTase) to alter developmental programs for therapeutic and biological use.